(8) Insurmountable antagonism is characterized by a decrease in the maximal agonist-induced response, which cannot be overcome by increasing the concentration of agonist. (6,7) For example, the action of angiotensin-1 receptor antagonists is prolonged by their long-lasting, induced-fit binding to their target receptor, reflected by insurmountable antagonism in in vitro experiments. (5) Ligand binding kinetics is presumed to play an important role in determining the time course of occupancy at the target receptor. (1−4) The time course of receptor occupancy has been suggested to have an important impact on the clinical properties of therapeutic ligands, including antipsychotics, where transient rather than continuous D 2R occupancy may be associated with a more favorable profile in terms of extrapyramidal side effects. D 2R antagonism or weak partial agonism is the common denominator of antipsychotic drugs, and D 2R agonism is a mainstay of Parkinson’s disease treatment. The dopamine D 2 receptor (D 2R) is a G protein-coupled receptor (GPCR) and an important pharmaceutical target. We propose a mechanism, supported by in silico modeling, whereby SV-III-130 initially binds reversibly to the D 2R, after which the drug-receptor complex undergoes a slow transition to a second ligand-bound state, which is dependent on secondary binding pocket integrity and irreversible during the time frame of our experiments. Our results suggest that the secondary binding pocket influences recovery from inhibition by the studied aripiprazole analogues.
Two mutations in the secondary binding pocket (V91A and E95A) of D 2R decreased antagonistic potency and increased recovery from SV-III-130 antagonism, whereas a third mutation (L94A) only increased recovery. Curve-shift experiments were consistent with competition between SV-III-130 and DA. Prolonging the coincubation time with SV-III-130 further diminished recovery. Two compounds containing 3- and 5-carbon linkers allowed for a similar extent of recovery from antagonism in the presence of 1 or 100 μM DA (>25 and >90% of control, respectively), whereas recovery was less prominent (∼20%) upon washout of the 4-carbon linker compound, SV-III-130, both with 1 and 100 μM DA. The aripiprazole analogues comprise an orthosteric and a secondary pharmacophore and differ by the length of the saturated carbon linker joining these two pharmacophores.
#Insurmountable antagonism series
Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in Xenopus oocytes to measure the kinetics of D 2R antagonism by a series of aripiprazole analogues, as well as the recovery of dopamine (DA) responsivity upon washout.
A solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D 2 receptor (D 2R).
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